Jie:LabNotes/CpgSeq/2008-7-31: Difference between revisions

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   37C 2h
   37C 2h
   MinElute column purify. Elute in 22ul EB.
   MinElute column purify. Elute in 22ul EB.
[[Image:20080801_after MmeI.jpg]]20080801_after MmeI


==step3. USER digestion==
==step3. USER digestion==
                                  x2
   DNA                    10ul        
   DNA                    10ul     20ul 
   USER                    3ul      
   USER                    3ul       6ul 
   total                  13ul           
   total                  13ul           


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   37C 10mins.
   37C 10mins.
   Minelute cloumn purify. Elute in 18ul H2O.
   Minelute cloumn purify. Elute in 18ul H2O.
[[Image:20080801_after USER and S1.jpg]]20080801_after USER and S1


==step5. end repair==
==step5. end repair==
                                      
                                     positive control
   Total                  25ul                      
   Total                  25ul           H2O 13ul           
   DNA                    17ul                       
   DNA                    15ul            2ul(20080801_spacer_107bp)           
   dNTP                  2.5ul           
   dNTP                  2.5ul          2.5ul
   dATP                  2.5ul                
   dATP                  2.5ul           2.5ul     
   10xendrepair buffer  2.5ul            
   10xendrepair buffer  2.5ul           2.5ul 
   enzyme                0.5ul        
   enzyme                0.5ul           0.5ul
    
   extra ATP(10mM)      2.5ul          2.5ul
 
   Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
   Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.


==step6. adapter ligation==
==step6. adapter ligation==
                            
                            
   total                      30ul       control          
   total                      30ul   positive control    negative control      
   DNA                        13ul       ddH2O 13ul             
   DNA                        13ul         13ul            ddH2O 13ul             
   100uM Solexa_1 adaptor    0.5ul        0.5ul  
   100uM Solexa_1 adaptor    0.5ul        0.5ul                0.5ul
   100uM Solexa_2 adaptor    0.5ul        0.5ul                 
   100uM Solexa_2 adaptor    0.5ul        0.5ul                0.5ul
   2xQuickLiage buffer        15ul          15ul  
   2xQuickLiage buffer        15ul          15ul                 15ul
   QuickLigase enzyme(NEB)      1ul          1ul    
   QuickLigase enzyme(NEB)      1ul          1ul                   1ul
 
   extra ATP(10mM)           2.5ul        2.5ul                2.5ul
 
   Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB.


  Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul H2O.


[[Image: 20080801_after endrepair and ligation.jpg]]


   PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.
   PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.


==step7. Nick-translation==
==step7. Nick-translation==
Line 92: Line 96:
                                                 x3
                                                 x3
   Nick-translated DNA          10ul             
   Nick-translated DNA          10ul             
   Solexa_PCR_up(10uM)          1ul           3ul  
   Solexa_PCR_up(10uM)          2ul           6ul  
   Solexa_PCR_lo(10uM)          1ul           3ul
   Solexa_PCR_lo(10uM)          2ul           6ul
   2xiProof master mix          25ul          75ul  
   2xiProof master mix          50ul          150ul  
   50x SYBG                    0.4ul         1.2ul
   50x SYBG                    0.8ul         2.4ul
   ddH2O                      12.6ul         37.8ul
   ddH2O                      35.2ul         106.6ul
   98C 30sec -> 13 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
   98C 30sec -> 12 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
 


[[Image:20080803_sequencing library.jpg]]20080803_sequencing library


Purify with Qiaquick column. Elute in 30ul EB.
Purify with Qiaquick column. Elute in 30ul EB.
dUTP250uM: 12.8ng/ul
yield: 22.9ng/ul(173nM)
dUTP100uM: 22.5ng/ul

Latest revision as of 16:57, 4 August 2008

sequencing library construction[edit]

2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40[edit]

 reaction system                                                x4         x3 
 H2O                                                43.2ul     172.8ul  518.4
 2x Master mix                                        50ul      200ul     600
 dUTP(1mM)                                             2ul        8ul      24
 AmpF6.3(10uM)                                         2ul        8ul      24
 AmpR6.3(10uM)                                         2ul        8ul      24
 50x SYBG I                                          0.4ul      1.6ul     4.8
 template(18.4ng/ul)                                 0.1ul      0.4ul     1.2
 Total                                               100ul      400ul     2400
 94C 3min -> 13cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 
 Bioneer column purification. Yield:167ng/ul x60ul

step2. Digestion with MmeI[edit]

                        dUTP 250uM,167.3ng/ul      x2 
 Total                               20ul      
 DNA                                  6ul          12  
 10X NEBuffer 4                       2ul           4 
 1mM SAM(fresh)                       2ul           4
 2U/ul Mme I                          8ul          16 
 ddH2O                                2ul           4 
 
 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h
 MinElute column purify. Elute in 22ul EB.

File:20080801 after MmeI.jpg20080801_after MmeI

step3. USER digestion[edit]

                                 x2
 DNA                    10ul      20ul   
 USER                    3ul       6ul  
 total                  13ul           

37C 12h.

step4. S1 nuclease digestion[edit]

                                   x2
 10 x S1 nuclease buffer:   2ul     4ul
 DNA after USER digestion: 13ul    26ul
 S1 nuclease (10U/ul):      1ul     2ul
 ddH2O                      4ul     8ul
 37C 10mins.
 Minelute cloumn purify. Elute in 18ul H2O.

File:20080801 after USER and S1.jpg20080801_after USER and S1

step5. end repair[edit]

                                   positive control
 Total                  25ul           H2O 13ul             
 DNA                    15ul            2ul(20080801_spacer_107bp)             
 dNTP                  2.5ul           2.5ul
 dATP                  2.5ul           2.5ul      
 10xendrepair buffer   2.5ul           2.5ul  
 enzyme                0.5ul           0.5ul
 extra ATP(10mM)       2.5ul           2.5ul 
 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.

step6. adapter ligation[edit]

 total                       30ul    positive control     negative control       
 DNA                         13ul          13ul            ddH2O 13ul            
 100uM Solexa_1 adaptor     0.5ul         0.5ul                 0.5ul
 100uM Solexa_2 adaptor     0.5ul         0.5ul                 0.5ul
 2xQuickLiage buffer         15ul          15ul                  15ul
 QuickLigase enzyme(NEB)      1ul           1ul                   1ul
 extra ATP(10mM)            2.5ul         2.5ul                 2.5ul
 Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul H2O.

File:20080801 after endrepair and ligation.jpg

 PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.

step7. Nick-translation[edit]

 set up the ligation system:
 Total                       20ul              
 DNA                         15ul               
 10x ThermoPol buffer         2ul          
 10mM dNTP                  0.4ul           
 1mg/ml BSA                   2ul            
 Bst polymerase(8U/ul)        1ul             
 65C for 25 minutes -> keep on ice.

step8. PCR of sequencig library[edit]

                                               x3
 Nick-translated DNA          10ul             
 Solexa_PCR_up(10uM)           2ul            6ul  
 Solexa_PCR_lo(10uM)           2ul            6ul
 2xiProof master mix          50ul          150ul  
 50x SYBG                    0.8ul          2.4ul
 ddH2O                      35.2ul         106.6ul
 98C 30sec -> 12 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.

File:20080803 sequencing library.jpg20080803_sequencing library

Purify with Qiaquick column. Elute in 30ul EB. yield: 22.9ng/ul(173nM)